PROJECTSUMMARY/ABSTRACT In most organisms, the genetic code, consisting of 64 triplets of nucleotides, encodes 20 amino acid building blocks used in the synthesis of proteins. The overall goal of the PI?s research program is to develop interdisciplinary tools to reprogram the genetic code to precisely probe and manipulate biological systems. Centraltoreprogrammingthegeneticcodeisourabilitytoaddnoncanonicalaminoacids(ncAAs)toproteinsof interest.TheoverallgoalofthisproposalistodevelopcellsabletobiosynthesizeandutilizencAAsandexplore theutilityortheseunnaturalorganismsinproteinevolutionandtherapydevelopment.Toachievethisgoal,the first research direction will focus on the generation of completely autonomous organisms with a variety of 21st aminoacids.Theprokaryoticandeukaryoticcellswiththe21staminoacidwillharborabiosyntheticpathwayand a bioorthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair for the new amino acid building block. The biosynthesis pathway of ncAAs will be obtained from other species or via metabolic repurposing. To site- specificallyincorporatethesencAAsintoproteins,wewillevolvebioorthogonalaaRS/tRNApairsandaddthem to the cells. The resulting organisms with a 21st amino acids will allow for the evolution of proteins with novel activities as well as the development of new therapies. To evolve novel or enhanced enzyme activity not accessiblebythe20canonicalaminoacids,alibraryofncAA-containingenzymemutantswillbegeneratedin the unnatural organisms and subjected to a fluorescence-activated cell sorting (FACS)-based or survival selection. The evolved ncAA-dependent enzymes can be used to prevent the unintended proliferation of genetically modified organisms or to prepare autotrophic vaccines. Next, we will explore the utility of these unnatural organisms with additional protein building blocks for therapeutic development. The prokaryotic and eukaryoticcellsabletobiosynthesizeandutilizeaminoacidswithbioorthogonalhandleswillbeusedtoproduce antibody variants with optimized therapeutic efficacy. Engineered immune cells with additional building blocks willallowfortheredirectionofthespecificityofchimericantigenreceptor(CAR)-immunecells,thusprovidinga new design strategy for switchable CAR-immune cells. Our efforts in this project will yield a collection of organisms with additional amino acids building blocks, and will result in versatile platforms for ncAA-based proteinevolutionortherapeuticproteinsthatcouldrevolutionizemodernmedicine.